KR101761194B1 - Composition Containing Paeonia Radix Alba Extract for Preventing and Treating Osseous Metabolic Disease - Google Patents

Abstract

The present invention relates to a composition for preventing or alleviating bone metabolic diseases comprising an extract of Veterinary Medicine as an active ingredient. The composition of the present invention inhibits osteoclast formation by inhibiting the expression of NFATc1 induced by RANKL in osteoclasts, And exhibits an effect of inhibiting bone resorption by osteoclasts. Therefore, it can be effectively used for the treatment of bone metabolic diseases through inhibition of osteoclast formation and inhibition of bone resorption. Specifically, bone metastatic cancer, solid tumor bone metastasis, musculoskeletal complication due to solid tumor metastasis, For the treatment of bone metabolic diseases such as hypercalcemia, multiple myeloma, primary bone tumors, osteoporosis, rheumatoid arthritis, degenerative arthritis, periodontal disease, inflammatory alveolar bone disorder, inflammatory bone resorption disease and Paget's disease And can be usefully used for prevention or treatment of bone metastasis of solid tumors such as breast cancer.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing and treating bone metabolic diseases,

The present invention relates to a composition for the prevention and treatment of bone metabolic diseases, which comprises a Viroxx extract. More particularly, the present invention relates to a composition for prevention and treatment of bone metabolic diseases, have.

Paeoniae Radix Alba is a medicinal herb that uses dried roots of buttercups and melons to gather and harvest blood, which is a nutrient for the liver, It is known that it has the efficacy of a salt-water-reducing effect (sweating 汗 汗), a sweat-stopping efficacy, and an efficacy that relaxes the heart and stops the pain. It has also been reported to be effective in various pain, such as abdominal pain, headache, menstrual irregularities, menstrual pain, seizures and pain in the limbs. The ingredients of the vinegar contain paeoniflorin and alkaloid paeonine as glycosides and also contain tannin resin benzoic acid. Paeoniflorin, a representative component of the veterinary medicine, was isolated, and various studies on anti-inflammatory action and stress ulcer prevention were reported. Antitumor effect, inhibition of food poisoning-induced bacterial growth inhibition, immunity enhancement effect, protection against radiation, skin effect, skin irritation, anti-inflammatory effect, anti- Whitening and wrinkle prevention effects and antioxidant effects against oxidative stress have been reported.

On the other hand, Korean Patent Registration No. 10-1235238 discloses that a veterinary drug inhibits cartilage cell damage and promotes growth of chondrocyte cells, thereby demonstrating the therapeutic effect of a degenerative disease.

Korean Patent Laid-Open Publication No. 10-2006-0078305 discloses a cosmetic composition containing extracts of yuguanpi, lily, bananas, white wax and white marlin having an inhibitory activity against elastase which hydrolyzes elastin fibers important for skin elasticity, Effect, cell proliferation effect, and anti-inflammatory activity, and has skin elasticity enhancement and anti-inflammatory effect.

Korean Patent Laid-Open No. 10-2006-0023884 has an antioxidative activity to protect the damage of nerve cells induced by cerebral ischemia because of the antioxidative activity of the pesticide extract. Therefore, the composition comprising the nerve cell extract is useful as a degenerative brain disease That is, it can be used for the prevention and treatment of stroke, stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick's disease and Creutzfeld-Jakob disease. However, the prevention and treatment of osteopathic diseases of Viroxx extract is not known.

On the other hand, the main constituent of bone is collagen and various proteins and hydroxyapatite minerals. Osteoclasts, which are cells that degrade bone matrix, are specialized cells that are responsible for bone resorption in bone remodeling process. They are formed from progenitor cells such as monocytes or macrophages through differentiation programs do. (Hadjidakis DJ, and Androulakis II, Ann. N. Y. Acad Sci., 1092: 385396, 2006). Bone metabolism and bone remodeling occur through a harmonious balance between the osteoblasts that make up the bone matrix and the osteoclasts that absorb the bones. Multinucleated osteoclasts are produced from hematopoietic stem cells through a series of processes including proliferation, differentiation, fusion, and activation. In osteoclast, osteoclasts are characterized by the conversion of mononuclear cells to pluripotent cells and the activity of TRAP (tartrate-resistant acid phosphatase). Therefore, when identifying osteoclast differentiated, osteoclast is stained with TRAP to observe TRAP- (Lee SE et al., Bone 30: 71-77, 2002).

Differentiation of osteoclasts by RANKL is further promoted in the presence of some inflammatory cytokines and chymocaine. The macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor kappa-B ligand (RANKL), which are produced from osteoblasts, are monocyte / macrophage differentiated from hematopoietic stem cells. It is an important cytokine that plays a role in differentiating precursor cells into osteoclasts. When RANKL produced in osteoblast cells binds to a receptor called RANK (receptor activator of nuclear factor kappa-B) on the surface of osteoclast precursor cells, intracellular signal transduction plays an important role in osteoclast differentiation, N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), mitogen-activated protein kinases (MAPKs) such as p38, and activation of NF-κB and Akt. RANKL regulates the expression of various transcription factors such as NF-κB, c-Fos, and NFAT, which act as positive modulators of osteoclast differentiation and production. When RANKL binds to RANK during osteoclastogenesis, c-Fos expression is increased. Expression Increased c-Fos binding to the promoter region of NFATc1 leads to the expression of NFATc1. NFATc1 then induces abrupt expression of NFATc1 during osteoclastogenesis induced by RANKL by binding to its promoter and increasing its expression by auto-amplification. The expression of constitutive active form of NFATc1 in osteoclast precursor cells induced osteoclastogenesis even in the absence of RANKL and that the NFATc1 deficient embryo cells were not differentiated into osteoclasts when treated with RANKL The results show that NFATc1 is the most important transcription factor that regulates osteoclast differentiation and production. NFATc1 induces expression of target genes such as TRAP (tartrate resistant acidic phosphatase), CtsK (Cathepsin K), and OSCAR (osteoclast-associated receptor), which play an important role in osteoclast differentiation and function. Recently, expression of ATP6v0d2 (d2 isoform of vacuolar H ⁺ -ATPase V0 domain) and DC-STAMP (dendritic cell-specific transmembrane protein) gene, which are known to regulate osteoclast fusion, have also been reported to be induced by NFATc1. Therefore, NFATc1 seems to be the most important regulator in all stages of RANKL-induced osteoclast differentiation, fusion, activation and production.

Korean Patent No. 10-1235238 Korean Patent Publication No. 10-2006-0078305 Korean Patent Publication No. 10-2006-0023884

Kim TJ, Shin JY. Korean oriental medicine keeping with our herbal (3). 1st ed. Seoul: Liyou. 2003: 154-157. Kim J. Enhancing Effect of Paeonia japonica, Houttuynia cordata, and Aster scaber Extracts on the Immunoreactivity in Vivo in Mice. Graduate School of Sookmyung Women's University. Ph.D Thesis, 2003. Aimi N, Inaba M, Watanabe M, Shibata S. Chemical studies on the oriental plant drugs. 23. Peoniflorin, a glucoside of Chinese paeony root. Tetrahedron. 1969; 25 (9): 1825-1838. Takagi K, Harada M. Pharmacological studies on herb paeony root. II. Anti-inflammatory effect, inhibitory effect on gastric juice secretion, preventive effect on stress ulcer, antidiuretic effect of paeoniflorin and combined effects with licorice component Fm 100. Yakugaku Zasshi. 1969; 89 (7): 887-892. Park HR, Jung U, Jeong I Y, Yee ST, Jo SK. Inhibition of Tumor Growth through Macrophage Activation by Polysaccharide Fraction from Peonia japonica (PJ-P). J Korean Soc Food Sci Nutr. 2003; 32 (1): 149-154. Yoon HJ, Go EB, Choi MS, Kim DI, Sung JS. Antioxidant Effect of Paeonia Japonica Extracts on Mouse Embryonic Fibroblast Cells. J Korean Obstet Gynecol. 2012; 25 (2): 78-88. Yang M, Xiao C, Wu Q, Niu M, Yao Q, Li K, Chen Y, Shi C, Chen D, Feng G, Xia C. Anti-inflammatory effect of Sanshuibaihu decoction associated with nuclear factor-κB and p38 MAPKa in collagen-induced arthritis in rat. J Ethnopharmacol. 2010; 127 (2): 264-273.

The present invention is to provide a composition for prevention and treatment of bone metabolic diseases, which comprises a Viroxx extract.

Specifically, the present invention provides a pharmaceutical composition for preventing or treating bone metabolic diseases, a food composition for preventing or improving bone metabolic diseases, and a health functional food composition containing an extract of Baekjak extract as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating bone metabolic diseases comprising an extract of Vaccinium myrcum as an active ingredient.

In one aspect of the invention, the bone metabolic disease is selected from the group consisting of bone metastatic cancer, solid tumor bone metastasis, musculoskeletal complications due to solid tumor metastasis, hypercalcemia due to malignancy, multiple myeloma, primary bone tumor, But are not limited to, osteoporosis, rheumatoid arthritis, degenerative arthritis, periodontal disease, inflammatory alveolar bone disease, inflammatory bone resorption disease and Paget's disease, but are not limited to, It can be used for diseases that cause pathological conditions.

In one embodiment of the present invention, the albedo extract comprises water, a lower alcohol having 1 to 4 carbon atoms, a mixed solvent of lower alcohol having 1 to 4 carbon atoms and water, acetone, ethyl acetate, chloroform, butyl acetate, The solvent may be extracted with a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, a lower alcohol having 1 to 4 carbon atoms and water, have.

In one embodiment of the present invention, the albedo extract exhibits an activity of inhibiting the differentiation or production of osteoclast, inhibiting the expression of NFATc1 transcription factor involved in osteoclast differentiation, and thus exhibiting the effect of preventing or treating bone metabolic diseases.

The present invention also provides a food composition for preventing or ameliorating a metabolic disease containing bone-marrow extract, or a health functional food.

In one aspect of the invention, the bone metabolic disease is selected from the group consisting of bone metastatic cancer, solid tumor bone metastasis, musculoskeletal complications due to solid tumor metastasis, hypercalcemia due to malignancy, multiple myeloma, primary bone tumor, But are not limited to, osteoporosis, rheumatoid arthritis, degenerative arthritis, periodontal disease, inflammatory alveolar bone disease, inflammatory bone resorption disease and Paget's disease, but are not limited to, May be used for the purpose of preventing or ameliorating a disease causing pathological conditions.

In one embodiment of the present invention, the albedo extract comprises water, a lower alcohol having 1 to 4 carbon atoms, a mixed solvent of lower alcohol having 1 to 4 carbon atoms and water, acetone, ethyl acetate, chloroform, butyl acetate, The solvent may be extracted with a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, a lower alcohol having 1 to 4 carbon atoms and water, have.

The present invention also provides an osteoclast differentiation inhibitor comprising an albedo extract as an active ingredient.

The composition of the present invention inhibits osteoclast formation by inhibiting the expression of RANKL-induced NFATc1 in osteoclasts and exhibits an effect of inhibiting osteoclast-induced bone resorption. Therefore, it can be effectively used for prevention and treatment of bone metabolic diseases through inhibition of osteoclast formation and inhibition of bone resorption. Specifically, bone metastatic cancer, solid cancer bone metastasis, musculoskeletal complications due to solid tumor metastasis, Bone metabolic diseases such as hypercalcemia, multiple myeloma, primary bone tumor, osteoporosis, rheumatoid arthritis, degenerative arthritis, periodontal disease, inflammatory alveolar bone disorder, inflammatory bone resorption disease and Paget's disease Can be used as a preventive or therapeutic agent for a cancer, and as an additive for a functional food. Especially, it can be useful for prevention or treatment of bone metastasis of solid cancer such as breast cancer.

FIG. 1 is a graph showing the results of cytotoxicity test on osteoclasts when PRAE was treated. FIG. Data represent the mean value of three experiments (± SD). NS: No significant difference
Figure 2 is a graph showing the effects of PRAE on osteoclast diversity. The data represent the mean value of three independent experimental results (± SD). NS: No significance, * P <0.05 and † P <0.01 vs control (0 ㎍ of PRAE)
FIG. 3 is a graph showing the results of real-time PCT analysis of the effect of PRAE on expression of various osteoclast marker genes. Data represent mean values of three independent experiments (± SD). * P <0.05, † P <0.01 vs control (0 ug / ml of PRAE).
FIG. 4 is a graph showing the effect of PRAE on RANKL-induced osteoclast signaling and transcription factor expression.

The present invention provides a composition for the prevention and treatment of bone metabolic diseases, which comprises a Viroxx extract.

The bauxite used in the present invention is pea lactiflor a) as root, which means that it is boiled and then dried.

The term &quot; extract &quot; used herein to refer to a veterinary drug includes not only the extraction result obtained by treating the veterinary extract with an extraction solvent, but also a veterinary medicine including a veterinary medicine (for example, pulverized) so that the veterinary medicine itself can be administered to an animal .

Various extracting solvents can be used in the case of obtaining the veterinary extract used in the composition of the present invention by treating the vinegar with an extraction solvent. Specifically, a polar solvent or a non-polar solvent can be used.

In one embodiment of the invention, suitable as a polar solvent is a mixture of (i) water, (ii) an alcohol (preferably methanol, ethanol, propanol, butanol, n-propanol, iso-propanol, n-butanol, Butoxyethanol or ethylene glycol), (iii) acetic acid, (iv) DMFO, and (v) dimethyl sulfoxide (DMSO).

In one embodiment of the present invention, suitable nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, Chlorobutane, benzene, diethyl ether, diisobutyl ether, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, Diethylsulfide, chloroform, dichloromethane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride and THF.

In one embodiment of the present invention, more specifically, the extraction solvent used in the present invention comprises (a) water, (b) an anhydrous or hydric alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, (e) ethyl acetate, (f) chloroform, (g) butyl acetate, (h) 1,3-butylene glycol, (i) hexane, and (j) diethyl ether. Specifically, the extract of the present invention is obtained by treating water, methanol, ethanol or a combination thereof with a vinegar.

As used herein, the term &quot; extract &quot; means that it is used in the art as a crude extract as described above, but broadly includes fractions obtained by further fractionating the extract. That is, the vile extract includes not only the extract obtained by using the above-mentioned extraction solvent, but also the addition of the purification process thereto. For example, fractions obtained by further fractionating the extract with the solvent described above, fractions obtained by passing the fraction through an ultrafiltration membrane having a constant molecular weight cut-off value, various chromatographies (size, charge, hydrophobicity or affinity And the fraction obtained through various further purification methods, such as the separation by the above-mentioned separation method, is also included in the extract of Veterinary Medicine of the present invention. The extract of the Viroxx extract used in the present invention can be prepared in a powder state by an additional process such as vacuum distillation and freeze drying or spray drying.

As used herein, the term &quot; bone metabolic disease &quot; refers to a disease that results in a physiologic pathology that is excessive bone resorption. The extract of the present invention can inhibit bone resorption by osteoclasts and thus can exhibit the therapeutic effect of bone metabolic diseases.

In one aspect of the present invention, examples of bone metabolic diseases include osteoporosis, osteopenia, Paget's disease, osteomyelitis, infectious lesions caused by bone loss, hypercalcemia, bone destruction due to rheumatism, joint destruction or osteopenia, osteoarthritis, Osteonecrosis accompanied by systemic lupus erythematosus or non-traumatic injuries or osteoporosis, osteodystrophy and solid tumors of bone, and specifically bone metastasis, cancer metastasis, cancer metastasis, traumatic injury, osteoporosis, sickle cell anemia, Bone metastatic cancer, solid tumor metastasis, musculoskeletal complications due to solid tumor metastasis, hypercalcemia due to malignant tumors, multiple myeloma, primary bone tumor, osteoporosis, rheumatoid arthritis, degenerative arthritis, periodontal disease, inflammatory Alveolar bone resorption disease, inflammatory bone resorption disease, and Paget's disease.

The composition of the present invention inhibits osteoclast differentiation and inhibits osteoclast-induced bone resorption by inhibiting the expression of NFATc1 (nuclear factor of activated T cells c1) transcription factor involved in osteoclast differentiation. Can be effectively used for the prevention and treatment of the above-mentioned bone metabolic diseases through inhibition of osteoclast differentiation and inhibition of bone resorption.

The pharmaceutical composition of the present invention may be orally administered or parenterally administered (for example, intravenous, subcutaneous, intraperitoneal, topical application, etc.) depending on the intended method, and the dose may be appropriately determined depending on the patient's body weight, The range varies depending on the condition, diet, administration time, method of administration, excretion rate, and severity of the disease.

According to one aspect of the present invention, there is provided an osteoclast differentiation inhibitor comprising an albedo extract as an active ingredient. In a specific example, the present inventors confirmed that the treatment of the extract of Veterinary Medicine with TRAP staining analysis and real-time PCR analysis inhibits the expression of NFATc1 transcription factor and inhibits osteoclast differentiation and production.

In one aspect of the present invention, the pharmaceutical composition may be formulated into a formulation selected from the group consisting of tablets, granules, pills, capsules, solutions, injections, ointments, suppositories and powders, but is not limited thereto.

Examples of the carrier, excipient and diluent which can be contained in the composition including the extract of Veterinary Medicine include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, aceitol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylidrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, for example starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The composition of the present invention is administered in a pharmaceutically effective amount.

The term "pharmaceutically effective amount &quot; as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will vary depending on the species and severity, age, sex, , Sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-drugs and other factors well known in the medical arts. However, for the desired effect, Or a pharmaceutically acceptable salt thereof, can be administered in an amount of 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg, once or several times a day, for an adult, The scope of the present invention is not limited thereto.

The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents for bone metabolic diseases, and can be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The term "individual &quot; in the present invention refers to all animals, including humans, who have already developed or are capable of developing a disease that can be prevented or treated through osteoclast differentiation or production inhibitory activity, The above diseases can be effectively prevented and treated.

In the present invention, &quot; treatment &quot; includes the partial cure, improvement and alleviation of bone metabolic disease symptoms as well as cure of bone metabolic diseases as a result of application of the pharmaceutical composition of the present invention to bone metabolic diseases.

In the present invention, &quot; prevention &quot; means that the pharmaceutical composition of the present invention is applied to a bone metabolic disease so as to inhibit or block the symptoms of bone metabolic diseases so that a bone metabolic disease does not occur in advance.

In the present invention, &quot; improvement &quot; is meant to include alleviation, prevention or treatment of symptoms.

In the present invention, the term &quot; active ingredient &quot; means an ingredient exhibiting activity alone or together with a carrier which is not active per se.

According to one aspect of the present invention, there is provided a food composition or a health functional food composition for preventing or improving bone metabolic diseases, which comprises an albedo extract as an active ingredient. The present invention can be used as a functional food or a food supplement, and the functional food can be appropriately used according to a conventional method in the art such as adding or processing a black lozenge extract as it is or using it.

In one aspect of the present invention, the health functional food may be formulated into a formulation selected from the group consisting of tablets, granules, pills, capsules, liquids and powders, but is not limited thereto.

The health functional food of the present invention may include the above-mentioned vile extract, and suitable food supplementary additives may be included.

The term "food-aid additive &quot; in the present invention means a component which can be added to foods in a supplementary manner, and it can be appropriately selected and used by those skilled in the art as being added to produce health functional foods of each formulation. A coloring agent and a filler, a pectic acid and a salt thereof, an alginic acid and a salt thereof, an organic acid, a protective colloid thickener, a pH adjuster, a stabilizer, a stabilizer and a stabilizer such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, A preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated drink, etc. However, the types of the food-aid additive of the present invention are not limited by the above examples.

The term "health functional food &quot; in the present invention refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and rings by using raw materials and components having useful functions in the human body. The term &quot; health functional food &quot; according to the present invention can be manufactured by a method commonly used in the art, for example, Unlike general drugs, it is advantageous in that there is no side effect that may occur when a drug is taken for a long time by using a food as a raw material, and in addition, The health functional food of the present invention is excellent in portability and is useful as an adjuvant for improving the prevention or improvement of bone metabolic diseases. It is possible.

The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the veterinary extract of the present invention may be contained in an amount of 0.01 to 5% by weight of the raw material composition when the food is prepared. In the case of health drinks, it may be added at a rate of 0.02 to 2 g or 0.3 to 1 g based on 100 mL. However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the amount can also be used in the above-mentioned range.

There is no particular limitation on the kind of the food or the health functional food. Examples of the food to which the extract can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, Dairy products, including soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, and includes food or health functional foods in a conventional sense.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. The following examples and experimental examples are provided to aid understanding of the present invention and are not intended to limit the scope of the present invention thereto.

&Lt; Materials and methods >

Preparation of the experiment 1. Extraction and preparation of the sample

The Paeoniae Radix Alba used in this experiment was purchased from the Korean Traditional Medicine University in Ilsan City in May 2012 and was identified through morphological features. The sample product (NNMBS-2012-066) was deposited at Wonkwang University, Respectively. 50 g of the pesticide was extracted with 50 ml of a 70% aqueous ethanol solution at 70 캜 for 2 hours and filtered. The filtrate was concentrated under reduced pressure to obtain 7.5 g of a pesticide extract. The 70% ethanol extract (NNMBS066, PRAE) was stored in Wonkwang University Natural Resources New Drug Standardization Research Materials Bank.

Experimental preparation 2. Osteoclast differentiation and culture

In this experiment, bone marrow cells from the femur and tibia of 6-8 week old C57BL / 6 male mice purchased from Orient Bio (Seongnam, South Korea) . The bone marrow cells were cultured in MEM-α (Minimum Essential Media Alpha, FBS, Hyclone, Rockford, IL, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, Rockford, IL, USA), 100 U / ml penicillin / 100 μg / ml streptomycin M-CSF at a concentration of 30 ng / ml was treated for 3 days in Hyclone medium at 37 ° C and 5% CO ₂ . Cells attached to the culture dish were separated by trypsin and centrifuged to collect the collected cells as bone marrow-derived macrophages (BMMs). BMMs were treated with 30 ng / ml of M-CSF and 100 ng / ml of RANKL in MEM-α medium supplemented with 10% FBS and 1% penicillin / tryptomycin for 4 days to produce osteoclasts. Induced differentiation into cells.

Preparation of experiment 3. Assessment of cytotoxicity and determination of PRAE concentration

Cytotoxicity was measured using the EZ-Cytox Enhanced Cell Viability Assay Kit from Itsbio (Korea) according to the manufacturer's instructions. First, BMMs cells were cultured and diluted to a concentration of 1 × 10 ⁴ cells / well. After adding 30 ng / ml of M-CSF and 100 ng / ml of RANKL, 96-well culture plates (Greiner bio- PRAE was treated at various concentrations (1, 2, 5, 10, 20 and 40 ㎍ / ㎖) and cultured for 2 days at 37 ℃ and 5% CO2. After incubation, 10 μl of EZ-Cytox reagent was added to each well and reacted at 37 ° C for 4 hours. After the reaction, the absorbance was measured at 450 nm using an ELISA reader (Tecan, Switzerland). Cytotoxicity values were expressed as percentage values relative to control (0 [mu] g / ml) without PRAE treatment.

Experimental Example 1. Measurement of PRAE effect on osteoclast differentiation and generation

BMMs cells were diluted in MEM-α medium supplemented with 30 ng / ml of M-CSF and 100 ng / ml of RANKL to a concentration of 1 × 10 ⁴ cells / well and dispensed into a 96-well culture plate PRAE was treated at various concentrations (1, 2, 5, 10, 20 and 40 ㎍ / ㎖) and then exchanged with fresh medium every 3 days for 4 days at 37 ℃ and 5% CO ₂ . After incubation, the cells were fixed with 10% formalin solution and methanol / acetone (1: 1) solution was added and the cells were treated for 1 minute and then dried at room temperature. 150 μl of TRAP substrate solution (pNPP in TRAP buffer, pH 5.2) was added to each well for analysis of TRAP solution to measure the total TRAP (tartrate resistant acidic phosphatase) activity in the culture plate, followed by reaction at room temperature for 30 minutes . After the reaction, 100 μl of the substrate solution was transferred to a new 96 cell culture plate, and 50 μl of 1N NaOH was added to each well to terminate the reaction. Absorbance was measured at 405 nm using an ELISA reader. After TRAP solution analysis, the culture plates were stained with TRAP for osteoclast identification. After washing three times with PBS, TRAP staining solution (Naphthol AS phosphate and Fast Red Violet dye in TRAP buffer, pH 5.2) was added to each well and reacted at room temperature until osteoclast staining. After reaction, TRAP (+) multinuclear cells (MNCs) with three or more nuclei were counted as microscopic oocytes.

Experimental Example 2. RNA isolation and real-time quantitative PCR (real-time PCR) analysis

To determine the degree of osteoclast differentiation, BMMs cells were treated with 30 ng / ml of M-CSF and 100 ng / ml of RNAKL, and 20 μg / ml of PRAE was added and cultured for 4 days. Cells cultured daily during the culture period were treated with a triazole reagent (Invitrogen, USA) to extract total RNA. 1 μg of total RNA was reverse transcribed using Maxima reverse transcriptase (Thermo Scientific, USA) and random primer to obtain cDNA. The synthesized cDNA and 10 pmol of each primer were added to the dataQuest SYBR Green qPCR Master mix (Affymetrix, USA) and real-time quantitative PCR analysis was performed using StepOne Plus ™ Real-Time PCR Systems (Applied Biosystems, USA). Expression levels of osteoclast differentiation factors (TRAP, CtsK, OSCAR, DC-STAMP, ATP6v0d2 and NFATc1) quantified by GAPDH were compared with the control group (0 μg / ml PRAE). The primers used at this time are shown in Table 1.

Experimental Example 3. Western blot analysis

The BMMs to 3 × 10 ⁵ cells / well concentration is to be diluted in M-CSF 30 ng / ㎖ MEM -α medium with the addition of the frequency divider 6 cell culture plate and then, 24 hours in a 37 ℃, 5% CO ₂ conditions Lt; / RTI &gt; The cells were treated with 100 ng / ㎖ of RANKL and 20 ㎍ / ㎖ of PRAE to measure the PRAE effect in RANKL - induced intracellular signal transduction. Or BMMs were diluted with a medium supplemented with M-CSF and RNAKL, followed by treatment with PRAE and culturing for 4 days. 1 mM phenylmethylsulfonyl fluoride (PMSF), protease-inhibitor cocktail (Roche, Germany) and phosphatase inhibitor tablets (Thermo Scientific, USA) The cultured cells were lysed with buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) and centrifuged to collect the supernatant It was used as seafood (total lysate). Protein concentration was measured by the Bradford method. 30 μg of total lysate was separated using 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred using Hybond ™ -PVDF (polyvinylidene fluoride membranes, GE-Healthcare Life Science, USA). Each membrane was blocked with 5% skin milk for one hour. The primary antibody was diluted 1: 1000 and allowed to react at room temperature for 2 hours. Secondary antibodies were HRP-conjugated IgG (1: 5000 dilution). The amount of protein expression was confirmed using an image analyzer (FluorChem E, PreoteinSimple, USA).

<Statistical Analysis>

The results were expressed as means ± SD from each of three experiments. Statistical analysis was performed with a one-way ANOVA test (SPSS 12.0, SPSS GmbH, Germany).

Table 1 above shows the nucleotide sequence of the primers used in the real-time PCR.

<Experimental Results>

1. Cytotoxicity of vinegar ethanol extract (PRAE)

To confirm the presence of cytotoxicity of PRAE on osteoclasts, PRAE was treated at 1, 2, 5, 10, 20, and 40 ㎍ / ㎖ in BMMs cells treated with M-CSF and RANKL, After culturing, cytotoxicity was measured. As a result, the cell viability of all experimental groups treated with PRAE was not significantly different from the control group without PRAE treatment. Therefore, it was confirmed that there was no cytotoxicity at the concentration to be used in the experiment (Fig. 1).

2. PRAE effect on osteoclast differentiation and generation

BMMs cells were treated with M-CSF and RNAKL to induce osteoclast differentiation and treated with PRAE at various concentrations (1, 2, 5, 10, 20, 40 ㎍ / ㎖) PRAE effect was measured. The osteoclasts were stained with TRAP and observed under a microscope. As the concentration of treated PRAE increased, the number of adult osteoclast, TRAP (+) MNCs was decreased (FIGS. 2A and 2B). In the experimental group treated with 1 ~ 2 ㎍ / ㎖ of PRAE, osteoclastogenesis was not significantly different from the control group without PRAE treatment. However, the production of osteoclast was significantly decreased in the experimental group treated with 5 ㎍ / ㎖ of PRAE, and the osteoclast was not generated from the experimental group treated with 20 ㎍ / ㎖ of PRAE. As a result of analysis of TRAP solution that can measure the total TRAP activity of single-, double-, and multi-cells expressing TRAP, the results of the TRAP solution showed that in the group treated with PRAE of 20 and 40 ㎍ / TRAP activity was decreased (FIG. 2C). These results show that PRAE inhibits osteoclast differentiation as well as osteoclast differentiation.

3. PRAE effect on expression of osteoclast differentiation factor

FIG. 2 shows that PRAE inhibits osteoclast differentiation and production. In order to verify this result in more detail, the expression levels of osteoclast differentiation factors (TRAP, CtsK, OSCAR, DC-STAMP, ATP6v0d2, NFATc1) . As a result, the expression level of all the osteoclast differentiation factors measured was significantly reduced in the cultured period in the group treated with PRAE at 20 / / ml compared to the control group not treated with PRAE (FIG. 3). In particular, expression of NFATc1, the most important transcription factor in all stages of osteoclast differentiation and production, was also reduced by PRAE. These results show that PRAE inhibits osteoclast differentiation and production by decreasing the expression of osteoclast differentiation factor and transcription factor.

4. PRAE effect on osteoclast signaling and transcription factor expression

To investigate the mechanism of PRAE - induced osteoclast differentiation and inhibition, we investigated RANKL - induced osteoclast signaling. BMMs cells were treated with M-CSF for 24 hours, and treated with RANKL for 5, 15, and 30 minutes to induce mitogen-activated protein kinases (MAPKs, ERK, JNK and p38) and IκBα activity. 20 ㎍ / ㎖ were simultaneously treated to investigate the MAPKs and IκBα activity. As a result, the degree of phosphorylation of ERK, JNK and p38 when treated with PRAE was not different from the control group without PRAE treatment. However, treatment of PRAE significantly inhibited phosphorylation of IκBα (FIG. 4A). In addition, the effect of PRAE on expression of c-Fos and NFATc1, the most important transcription factors for osteoclast differentiation and production, was measured (Fig. 4B). The expression of c-Fos was gradually increased when RANKL was treated, and it was most expressed after 24 hours. However, the expression of c-Fos was suppressed at all when treated with PRAE. Expression of NFATc1 was rapidly increased in the control group from 2 days, and the expression level was maintained even after 4 days. However, when treated with PRAE, it increased slightly after 2 days and decreased gradually. These results suggest that PRAE inhibits the expression of NFATc1 by inhibiting IκBα phosphorylation and c-Fos expression in RANKL-induced osteoclast differentiation. Thus, the transcription of many genes regulated by NFATc1 was inhibited, resulting in decreased osteoclast differentiation and production.

<Conclusion>

In the present invention, it was confirmed that the albedo extract (PRAE) showed an inhibitory effect on osteoclast differentiation and production induced by RANKL (FIG. 2). As a result of treatment with 20 μg / ml of PRAE, the activity of MAPKs (ERK, JNK, p38) remained unchanged, but the activity of IκBα was inhibited and the expression level of c-Fos was decreased. It was observed that the signal expression of the final NFATc1 was abruptly reduced by this signal suppression (Fig. 4). Also, it was confirmed that the expression amount of mRNA of NFATcl was reduced during osteoclast differentiation by PRAE (Fig. 3). The expression of osteoclast differentiation factors (TRAP, CtsK, OSCAR, DC-STAMP, ATP6v0d2) under the control of the transcription factor NFATcl was inhibited during the osteoclast differentiation process due to the reduction of expression of NFATc1 by PRAE. (Fig. 3).

Claims (8)

Wherein the extract of Veterinary Medicine contains water, a lower alcohol having 1 to 4 carbon atoms, a lower alcohol having 1 to 4 carbon atoms and water, acetone, ethyl acetate, chloroform, butyl acetate, 1,3 - bone metastatic cancer, solid tumor bone metastasis, musculoskeletal complications due to solid tumor metastasis, hypercalcemia due to malignant tumors, characterized by being extracted with a solvent selected from the group consisting of butylene glycol, hexane and diethyl ether. A pharmaceutical composition for the prevention or treatment of a bone metabolic disease selected from the group consisting of hyperlipidemia, multiple myeloma, primary bone tumor, osteoporosis, periodontal disease, inflammatory alveolar bone disorder, inflammatory bone resorption disease and Paget's disease.
delete delete The method according to claim 1,
A pharmaceutical composition for the prevention or treatment of bone metabolic diseases characterized by inhibiting the differentiation or production of osteoclasts and the expression of NFATc1 transcription factor involved in osteoclast differentiation.
Wherein the extract of Veterinary Medicine contains water, a lower alcohol having 1 to 4 carbon atoms, a lower alcohol having 1 to 4 carbon atoms and water, acetone, ethyl acetate, chloroform, butyl acetate, 1,3 - bone metastatic cancer, solid tumor bone metastasis, musculoskeletal complications due to solid tumor metastasis, hypercalcemia due to malignant tumors, characterized by being extracted with a solvent selected from the group consisting of butylene glycol, hexane and diethyl ether. A food composition for preventing or improving a bone metabolic disease selected from the group consisting of hyperlipidemia, multiple myeloma, primary bone tumor, osteoporosis, periodontal disease, inflammatory alveolar bone disorder, inflammatory bone resorption disease and Paget's disease.

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